This application is filed pursuant to 35 U.S.C. xc2xa7371 as a U.S. National Phase Application of International Application No. PCT/JP99/07286 filed Dec. 24, 1999, which claims priority from GB 9828704.8 filed Dec. 24, 1998.
This invention relates to a novel IKK kinase protein, IKK3, nucleotides coding for it, vectors and host cells containing the same and methods for screening for modulators of said IKK3 protein for treatment of conditions involving inflammation.
The transcription factor NF-kB controls the activation of various genes in response to pathogens and pro-inflammatory cytokines. Thus, for example, NF-kB is activated by various kinds of stimulation including tumour necrosis factor alfa (TNF alfa) and interleukin-1 (IL-1), bacterial LPS, viral infection, antigen receptor cross-linking of T and B cells, calcium ionophores, phorbol esters, UV radiation and free radicals (for reviews, see Varma et al., 1995, Genes Dev., 9, 2723-2735; Baueurerle and Baltimore, 1996, Cell, 87, 13-20), (see FIG. 2). NF-kB in turn controls the activation of various genes in response to these stimuli. Activation of these various genes in turn may result in the production of cytokines, chemokines, leukocyte adhesion molecules, hematopoietic growth factors and may also effect development and cell death as well as cell survival (see FIG. 1). Specifically, the transcription factor NF-kB controls the activation of various genes in response to pathogens and pro-inflammatory cytokines. The NF-kB activity is regulated through interaction with specific inhibitors, IkBs. Upon cell stimulation, the IkBs are rapidly phosphorylated and then undergo ubiquitin-mediated proteolysis, resulting in the release of active NF-kB (Baldwin, 1996, Annu. Rev. Immunol., 14, 649-681; Baueurerle and Baltimore, 1996, Cell, 87, 13-20), (see FIG. 2). It has been reported that the 700 kDa complex specifically phosphorylated IkBxcex1 at S32 and S36 (Chen et al., 1996, Cell, 84, 853-862).
Several groups found. that two kinases termed IKK1 and IKK2 (also known as IKKxcex1 and IKKxcex2), were the subunits of the kinase complex. The groups showed that the IKKs immunoprecipitates, derived from the TNFxcex1 or IL-1 stimulated cells are able to phosphorylate IkB in vitro. In addition to these observations, two groups reported that IKK1 and IKK2 purified from insect cells are able to phosphorylate IkB in vitro. These results suggested that IKK directly phosphorylates IkBs. The over expression of anti-sense IKK1, kinase-inactive IKK1 or IKK2 resulted in the inhibition of NF-kB activation mediated by TNFxcex1 and IL-1. These results suggest that IKKs are critical kinases in the NF-kB activation pathway (May and Ghosh, 1998, Immunol. Today 19, 80-88; Stancovski and Baltimore, 1997, Cell, 91, 299-302). It has, however, not been understood how upstream signals are transmitted to the kinase complex, or whether different kinase complexes might exist to phosphorylate distinct IkBs.
NEMO (NF-kB essential modifier) and IKKxcex3 (human homologue of the mouse NEMO) were isolated from purified IKK complex, and the inhibition of NEMO/IKKxcex3 gene expression impaired the cytokine induced NF-kB activation via IKK1 and IKK2. In NEMO deficient cells, smaller complexes of Mr 3,000-4,000 are formed, though the normal complex is Mr 7,000-9,000, suggesting that NEMO/IKKxcex3 physically link IkB kinase to upstream activators (Scheidereit, Nature, 1998, 395, 225-226).
The IKK-complex-associated protein (IKAP) was isolated from the IKK complexes. IKAP binds to IkB kinases and NIK and the complex, containing three kinases, leads to the maximum phosphorylation of IkB as compared to the complex containing one or two kinases. Accordingly, IKAP may act as scaffold proteins that link NIK or other molecules to IKK1 and IKK2 (Scheidereit, Nature, 1998, 395, 225-226). Accumulating evidence suggests that the IKK complex consists of several essential molecules, however, the molecular mechanisms that control the signalling complex were not well understood. Therefore, further association molecules were needed to complete the picture.
KIAA0151 was originally isolated from the KG-1 cDNA library (Nagase et al., 1995, DNA Res, 2, 167-174). KIAA0151 was identified as a potential Ser/Thr kinase, however, the importance of the molecule was not recognised. We have now found that KIAA0151 is similar to IKK1 and IKK2 using a computer homology analysis. KIAA0151, renamed IKK3, has a 21% homology with IKK1 and 23% with IKK2. IKK3 was able to phosphorylate IkB family proteins and directly phosphorylate IkB in vitro. The over expression of IKK3 leads to the activation of various inflammatory genes, such as IL-8, IL-6 and RANTES. These genes contain the NF-kB site in the gene regulation region. We know that IKK3 has an effect on IL-8 expression in Hela cells and also that IKK3 phosphorylates NF-kB. Moreover, it is known that the NF-kB site has an important role in IL-8 regulation. Our results suggest a correlation between IKK3 and the NF-kB site of the IL-8 promoter that has previously been identified as an endogenous NF-kB binding site, further suggesting that IKK3 plays an important role in controlling the NF-kB site of the IL-8 promoter. Specifically we have shown that IKK3 transactivates the IL-8 gene via the NF-kB binding to a site in the IL-8 promoter. These results lead to the conclusion that IKK3 is an important regulator of IL-8 gene regulation and thus activates genes that are important for the inflammatory diseases (see Table 1 below).
Using a computer homology analysis, we have now found that KIAA0151 is similar to IKK1 and IKK2. Importantly, recent experimental evidence has shown that IKK3 specifically controls various inflammatory genes, such as IL-8, IL-6 and RANTES. Moreover, IKK3 has been shown to phosphorylate various IkBs and directly phosphorylate TRIP9 (human IkBxcex2). IKK3 has therefore been shown to have a specific role in the control of inflammation.
Accordingly this invention provides a novel kinase protein, IKK3.
Nucleotide sequence analysis of IKK3 reveals a 2148 bp open reading frame which encodes 716 amino acid protein (FIG. 3). This deduced protein sequence shares many of the characteristics of IKK1 and IKK2. (see FIG. 5).
One aspect of the invention therefore provides an isolated IKK3 kinase protein or a variant thereof. The amino acid sequence of this isolated IKK3 kinase protein is shown in FIG. 3.
Included within the invention are variants of the IKK3 kinase protein. Such variants include fragments, analogues, derivatives and splice variants. The term xe2x80x9cvariantxe2x80x9d refers to a protein or part of a protein which retains substantially the same biological function or activity as IKK3.
Fragments can include a part of IKK3 which retains sufficient identity of the original protein to be effective for example in a screen. Such fragments may be probes such as the ones described hereinafter for the identification of the full length protein. Fragments may be fused to other amino acids or proteins or may be comprised within a larger protein. Such a fragment may be comprised within a precursor protein designed for expression in a host. Therefore, in one aspect the term fragment means a portion or portions of a fusion protein or polypeptide derived from IKK3.
Fragments also include portions of IKK3 characterised by structural or functional attributes of the protein. These may have similar or improved chemical or biological activity or reduced side-effect activity. For example, fragments may comprise an alpha, alpha-helix or alpha-helix-forming region beta sheet and beta-sheet-forming region, turn and turn-forming regions, coil and coil-forming regions, hydrophilic regions, hydrophobic regions, amphipathic regions (alpha or beta), flexible regions, surface-forming regions, substrate binding regions and regions of high antigenic index.
Fragments or portions may be used for producing the corresponding full length protein by peptide synthesis.
Derivatives include naturally occurring allelic variants. An allelic variant is an alternate form of a protein sequence which may have a substitution, deletion or addition of one or more amino acids, which does not substantially alter the function of the protein. Derivatives can also be non-naturally occurring proteins or fragments in which a number of amino acids have been substituted, deleted or added. Proteins or fragments which have at least 70% identity to IKK3 are encompassed within the invention. Preferably, the identity is at least 80%, more preferably at least 90% and still more preferably at least or greater than 95% identity for example 97%, 98% or even 99% identity to IKK3.
Analogues include but are not limited to precusor proteins which can be activated by cleavage of the precursor portion to produce an active mature protein or a fusion with a compound such as polyethylene glycol or a leader/secretory to aid purification.
A splice variant is a protein product of the same gene, generated by alternative splicing of mRNA, that contains additions or deletions within the coding region (Lewin N (1995) Genes V Oxford University Press, Oxford, England). The present invention covers splice variants of the IKK3 kinase protein that occur naturally and which may play a role in the control of inflammation.
The protein or variant of the present invention may be a recombinant protein, a natural protein or a synthetic protein, preferably a recombinant protein.
A further aspect of the invention provides an isolated and/or purified nucleotide sequence which encodes a mammalian IKK3 protein as described above, or a variant thereof. Also included within the invention are anti-sense nucleotides or complementary strands.
Preferably, the nucleotide sequence encodes a rat or human IKK3 protein. The nucleotide sequence preferably comprises the sequence of the coding portion of the nucleotide sequence shown in FIG. 4.
A nucleotide sequence encoding an IKK3 protein of the present invention may be obtained from a cDNA or a genomic library derived from the human fetus Marathon-Ready cDNA (Clonetech).
The nucleotide sequence may be isolated from a mammalian cell (preferably a human cell), by screening with a probe derived from the rat, murine or human IKK3 sequence, or by other methodologies known in the art such as preliminary chain reaction (PCR) for example on genomic DNA with appropriate oligonucleotide primers derived from or designed based on rat or human IKK3 sequence and/or relatively conserved regions of known IKK3 proteins. A bacterial artificial chromosome library can be generated using rat or human DNA for the purposes of screening.
The nucleotide sequence of the present invention may be in form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA and synthetic DNA. The DNA may be double-stranded or single-stranded, and if single-stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the IKK3 protein or variant thereof may be identical to the coding sequence set forth in FIG. 4, or maybe a different coding sequence which as a result of the redundancy or degeneracy of the genetic code, encodes the same protein as the sequences set forth therein.
A nucleotide sequence which encodes an IKK protein may include:
a coding sequence for the full length protein or any variant thereof;
a coding sequence for the full length protein or any variant thereof, and
additional coding sequence such as a leader or secretory sequence or a pro-protein sequence: a coding sequence for the full length protein or any variant thereof (and optionally additional coding sequence) and non-coding sequences, such as intrans or non-coding sequences 5xe2x80x2 and/or 3xe2x80x2 of the coding sequence for the full length protein. The invention also provides nucleotide variants, analogues, derivatives and fragments which encode IKK3. Nucleotides are included which preferably have at least 70% identity over the entire length to IKK3. More preferred are those sequences which have at least 80% identity over their entire length to IKK3. Even more preferred are polynucleotides which demonstrate at least 90% for example 95%, 97%, 98% or 99% identity over their entire length to IKK3.
The present invention also relates to nucleotide probes constructed from the nucleotide sequence of an IKK protein or variant thereof. Such probes could be utilised to screen a cDNA or genomic library to isolate a nucleotide sequence encoding an IKK3 protein. The nucleotide probes can include portions of the nucleotide sequence of the IKK3 protein or variant thereof useful for hybridising with mRNA or DNA in assays to detect expression of the IKK3 protein or localised its presence on a chromosome using for example flourescence in situ hybridisation (FISH).
The nucleotide sequences of the invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the protein of the present invention such as hexa-histadine tag or hemagglutinin (HA) tag, Myc-tag, T7-tag, double MYC-tag, double HA-tag and double T7-tag expression vectors or allows determination in screening assays of effective blockage of IKK3 or it""s modulation.
Nucleotide molecules which hybridise to IKK3 or to complementary nucleotides thereto also form part of the invention. Hybridisation is preferably under stringent hybridisation conditions. One example of stringent hybridisation conditions which is sometimes used is where attempted hybridisation is carried out at a temperature of from about 35xc2x0 C. to about 65xc2x0 C. using a salt solution which is about 0.9 mol. However, the skilled person will be able to vary such conditions as appropriate in order to take into account variables such as probe length, base composition, type of ions present etc. The nucleotide sequence of the present invention may be employed for producing the IKK3 protein or variant thereof by recombinant techniques. Thus, for example the nucleotide sequence may be included in any one of a variety of expression vehicles or cloning vehicles, in particular vectors or plasmids for expressing a protein, such vectors include chromosomal, non-chromosomal and synthetic DNA sequences. Examples of suitable vectors include derivatives of bacterial plasmids: phage DNA: yeast plasmids; vectors derived from combinations of plasmids and phage DNA and viral DNA. However, any other plasmid or vector may be used as long as it is replicable and viable in the host.
More particularly, the present invention also provides recombinant constructs comprising one or more of the nucleotide sequences as described above. The constructs comprise an expression vector, such as a plasmid or viral vector into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment the construct further comprises one or more regulatory sequences to direct messenger mRNA synthesis, including, for example a promoter operably linked to the sequence. Suitable promoters include: CMV, LTR, or SV40 promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector may contain an enhancer and a ribosome binding site for translation initiation and transcription terminator.
Large numbers of suitable vectors and promoters/enhancers, will be known to those of skill in the art, but any plasmid or vector, promoter/enhancer may be used as long as it is replicable and functional in the host.
Appropriate cloning and expression vectors for use with prokaryotic and eurkaryotic hosts include mammalian expression vectors, insect expression vectors, yeast expression vectors, bacterial expression vectors and viral expression vectors and are described in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, N.Y., (1989). The vector may also include appropriate sequences for selection and/or amplification of expression. For this the vector will comprise one or more phenotypic selectable/amplifiable markers, such markers are also well known to those skilled in the art.
In a further embodiment, the present invention provides host cells capable of expressing a nucleotide sequence of the invention, the host cell can be, for example, a higher eukaryotic cell, such as mammalian cell or a lower eukaryotic cell, such as a yeast cell or a prokaryotic cell such as a bacterial cell. Suitable prokaryotic hosts for transformation include E-coli. Other examples include viral expression vectors, insect expression systems and yeast expression systems.
Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
The IKK3. protein is recovered and purified from recombinant cell cultures by methods known in the art, including ammonium sulfate or ethanol precipitation, acid extraction, and ion or cation exchange chromatography, phosphocellulose chromatography and lecitin chromatography. Protein refolding steps may be used, as necessary, in completing configuration of the mature protein. Finally high performance liquid chromatography (HPLC) can be employed for final purification steps.
The proteins and nucleotide sequences of the present invention are preferably provided in an isolated form. The term xe2x80x9cisolatedxe2x80x9d means that the material is removed from its original environment e.g. the naturally-occurring nucleotide sequence or protein present in a living animal is not isolated, but the same nucleotide sequence or protein, separated from some or all of the materials it co-exists within the natural system, is isolated. Such nucleotide sequence could be part of a vector and/or such nucleotide sequence or protein could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. The proteins and nucleotide sequences of the present invention are also preferably provided in purified form, and preferably are purified to at least 50% purity, more preferably about 75% purity, most preferably 90% purity or greater such as 95%, 98% pure.
The present invention also provides antibodies specific for the IKK3 protein. The term antibody as used herein includes all immunoglobulins and fragments thereof which contain recognition sites for antigenic determinants of proteins of the present invention. The antibodies of the present invention may be polyclonal or preferably monoclonal, may be intact antibody molecules or fragments containing the active binding region of the antibody, e.g. Fab or (Fab)2. The present invention also includes chimaeric, single chain and humanised antibodies and fusions with non-immunoglobulin molecules. Various procedures known in the art may be used for the production of such antibodies and fragments.
The proteins, their variants especially fragments, derivatives, or analogues thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto. Antibodies generated against the IKK3 protein can be obtained by direct injection of the polypeptide into an animal, preferably a non-human. The antibody so obtained will then bind the protein itself. In this manner, even a sequence encoding only a fragment of the protein can then be used to generate antibodies binding the whole native protein. Such antibodies can be used to locate the protein in tissue expressing that protein.
The antibodies of the present invention may also be of interest in purifying an IKK3 protein and accordingly there is provided a method of purifying an IKK3 protein or any portion thereof which method comprises the use of an antibody of the present invention.
The present invention also provides methods of identifying modulators of the IKK3 protein. Screens can be established for IKK3 enabling large numbers of compounds to be studied. High throughput screens may be based on 14C guanidine flux assays and flourescence based assays as described in more detail below. Secondary screens may involve electrophysiological assays utilising patch clamp technology or two electrode voltage clamps to identify small molecules, antibodies, peptides, proteins or other types of compounds that inhibit, block, or otherwise interact with the IKK3 protein. Tertiary screens may involve the study of the modulators in well characterised rat and mouse models of inflammation. These models of inflammation include, but are not restricted to inflammatory models (murine) atopic dermatitis models (murine and rat), repeated-induced type dermatitis model (murine) and allergic asthma models (murine and guinea pig). For example, screens may be set up based on an in vitro phosphorylation system using bacterially expressd IKK3 proteins (see Example 5 and FIG. 12). This system may be used to screen for modulators of the IKK3 kinase activity and then subsequently testing the effect of potential modulators of IKK3 on gene expression, specifically the expression of IL-8, IL-6 and RANTES using cell based assay systems. Finally the efficacy of these modulators in relation to inflammatory or allergic diseases may be tested on models of inflammation.
The invention therefore provides a method of assaying for a modulator comprising contacting a test compound with the IKK3 protein and detecting the activity or inactivity of the IKK3 protein. Preferably, the methods of identifying modulators or screening assays employed transformed host cells that express the IKK3 protein. Typically, such assays will detect changes in the activity of the IKK3 protein to the test compound, thus identifying modulators of the IKK3 protein.
In general, a test compound is added to the assay and its effect on IKK3 is determined or the test compound""s ability to competitively bind to the IKK3 is assessed. Test compounds having the desired effect on the IKK3 protein are then selected.
IL-8, IL-6 and RANTES are involved in diseases involving inflammation and allergies. Specifically, asthma, atopic dermatitis, arthritis, rheumatoid arthritis, systemic lupus erythematosus, LPSxe2x80x94induced contact dermatitis, glomerulonephritis, gout and other inflammation-related diseases.
The invention therefore provides a modulator of a protein or a variant thereof as described above identifiable by a method described above for use in therapy. The invention further provides use of a modulator of an IKK3 protein optionally identifiable by a method described above for the manufacture of an anti-inflammatory medicament. Moreover the invention provides a method of treatment which comprises administering to a patient an effective amount of a modulator of a protein as described above. More specifically, the invention provides a method of treating diseases related to inflammation, such as asthma, atopic dermatitis, arthritis, rheumatoid arthritis, systemic lupus erythematosus, LPSxe2x80x94induced contact dermatitis, glomerulonephritis and gout.
Complementary or anti-sense strands of the nucleotide sequences as herein above defined can be used in gene therapy. For example, the cDNA sequence of fragments thereof could be used in gene therapy strategies to down regulate the IKK3 protein. Anti-sense technology can be used to control gene expression through triple-helix formation of anti-sense DNA or RNA, both of which methods are based on binding of a nucleotide sequence to DNA or RNA.
A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the product of the sodium channel. The anti-sense RNA oligonucleotide hybridises to the messenger RNA in vivo and blocks translation of the messenger RNA into the IKK3 protein.
The regulatory regions controlling expression of the IKK3 protein could be used in gene therapy to control expression of a therapeutic construct in cells expressing the IKK3 protein.